GOALTo demonstrate benefits of UPLC® separations

in HILIC mode for the glycans’ linkage analysis

of the released 2-AB labeled N-linked glycan

pool of fetuin, sequentially digested by

exoglycosidase array.

BAckGrOundMany biotherapeutics are glycosylated.

Glycoprotein characterization includes glycan

profiling, which is an important step in the

process of development and production of

biopharmaceutical proteins.

The released glycan pool is of a great complexity

due to branching and linkage isomers. Such

structural heterogeneity of glycans is attributed

to their biological role and requires thorough

characterization for glycobiology applications

and regulatory purposes.

At the same time, glycan complexity often

hinders complete structural elucidation by

conventional HPLC methods since many species

remain unresolved. Entire oligosaccharide

characterization is incomplete if structural or

linkage isomers cannot be closely monitored in

order to maintain consistency and high quality

during therapeutic manufacturing.

UPLC Separation of 2-AB Labeled Fetuin Glycans Removed by Exoglycosidase

Figure 1. Workflow of the enzymatic array digestion and the structures of the released bovine fetuin N-glycans.

In glycan analysis, uPLc delivers a clear advantage for resolving coeluting positional isomers, which are often difficult to separate with conventional HPLc.

UltraPerformance Liquid Chromatography® (UPLC) in hydrophilic interaction

chromatography (HILIC) separation mode has become a routine and widely-

recognized technique for rapid, efficient, sensitive, and reproducible analysis of

2-aminobenzamide (2-AB) labeled glycans. UPLC Glycan columns packed with

sub-2-µm particle sorbent offer significant improvements in separation power

and allow enhanced resolution of complex glycans.

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THe sOLuTIOnTo compare resolving capabilities of HPLC and UPLC

systems, a polysaccharide ladder of the released

bovine fetuin N-glycan was created by a sequential

digestion using enzyme array (Figure 1). Each

monosaccharide was released based on the enzyme

specificity (Figure 2).

LC experiments were run on a Waters ACQUITY UPLC®

System with fluorescence detection (FLR) and an

ACQUITY UPLC BEH Glycan Column (2.1 x 150 mm,

1.7 µm). The Waters Alliance® 2695 HPLC System

with a TOSOH Bioscience TSKgel Amide-80 Column

(2.0 x 150 mm, 3.0 µm) was used as the default HPLC

method. The structures of the digested glycans were

confirmed by glucose units (GU) values and specificity

of the applied enzyme.

The top chromatograms represent the undigested total

N-glycan pool, and the remaining chromatograms

correspond to the sequential digestion of the N-glycan

pool with the exoglycosidase enzymes indicated.

The first enzyme applied was Arthrobacter Ureafaciens

sialidase, which releases sialic acids. This step yields

two linkage isomers A3G(3,4,4)3 and A3G(4,4,4)3,

which are hard to resolve under HPLC conditions.

As highlighted on the chromatogram, these 2-AB

labeled isomers were separated using the 1.7-µm

ACQUITY UPLC BEH Glycan Column on the UPLC

system, but coeluted on the 3.0-µm HPLC column.

Such an improvement in resolution is due to the higher

peak capacity of sub-2-µm particle packing.

Method transfer between HPLC and UPLC is straight-

forward, since selectivity of neutral and siaylated

glycans in both columns are comparable under the

conditions used for this study.

summArySide-by-side comparisons of UPLC and HPLC methods of the fetuin N-glycan digest,

obtained by exoglycosidase array, illustrates the suitability of UPLC separations for

glycan identification. The results demonstrate UPLC’s clear advantage for resolving

coeluting positional isomers, which are often difficult to separate with a conven-

tional HPLC column and system.

This highly efficient and fast UPLC separation of glycans meets regulatory

obligations as a part of complete glycoprotein characterization, and conveniently

offers users a seamless HPLC-to-UPLC method transfer.

Figure 2. Sequential glycan removal from bovine fetuin. Linkage isomers not separated by HPLC are well-resolved using UPLC. Both UPLC and HPLC systems used solvent A: 100 mM Ammonium Formate, pH 4.5, solvent B: Acetonitrile; 72 to 57% B gradient in 45 min gradient time. Flow rate was 0.5 mL/min on the ACQUITY UPLC System and 0.45 mL/min on the Alliance 2695 HPLC System Figure 2. Sequential glycan removal from bovine fetuin. Linkage isomers not separated by HPLC are well-resolved using UPLC. Both UPLC and HPLC systems used solvent A: 100 mM Ammonium Formate, pH 4.5, solvent B: Acetonitrile; 72 to 57% B gradient in 45 min gradient time. Flow rate was 0.5 mL/min on the ACQUITY UPLC System and 0.45 mL/min on the Alliance 2695 HPLC System.